[unreadable] Non-human primates infected with Mycobaterium tuberculosis (M. tb.) and M. bovis develop disease and are thus not suitable for use in breeding or research projects. Chronically infected carriers are difficult to diagnose and present significant problems for maintaining colonies of healthy, high-quality animals for biomedical research. Currently, the main methods for screening are the tuberculin skin test and a blood cell stimulation assay. These conventional diagnostic systems have limitations due to problems with specificity and sensitivity. In addition, animals with latent infection (or low-level persistent infection) are difficult to identify by these methods. Accordingly, the objective of this R24 grant proposal is to develop novel immunoassays that are sensitive, specific, rapid, and economical for detecting infection of non-human primates with mycobacteria. The hypothesis is that a combination of multiple tests, based on conventional and novel immunoassay methods, will accurately and reliably determine M.tb./M. bovis infection of nonhuman primates. This project builds on recent advances in (1) knowledge of mycobacteria genomics, proteomics, and immunology and (2) novel diagnostic instrumentation for infectious disease. This project takes a two-pronged approach that utilizes experimentally inoculated macaques to characterize antigenspecific immune responses and naturally infected macaques to assess immunoassay performance. The following Specific Aims will develop and implement these assays: (1) to identify M. tb. proteins that are targets of humoral and cell-mediated immune responses in non-human primates experimentally inoculated with M. tb., (2) to develop and optimize novel multiplex immunoassays for detecting both humoral and cell-mediated immune responses targeted against the antigens identified in Specific Aim 1, (3) to test these novel multiplex and cell-based immunoassays in experimentally inoculated non-human primates at acute and latent stages of infection, and (4) to evaluate these immunoassays in naturally infected animals (i.e., during quarantine and in outbreaks in captive animals). The novel immunoassay methods developed in this proposal will be essential for establishing and maintaining specific-pathogen-free (SPF) colonies of nonhuman primates for optimal use in biomedical research, including the use of macaques for AIDS research. [unreadable] [unreadable] [unreadable]